Candida albicans is the most common fungal pathogen of humans, and causes infections that range from oral and vaginal candidiasis to life-threatening candidemia and invasive disease. Our broad objectives are to identify pathogen virulence mechanisms, prospective drug targets, and drug resistance mechanisms through large-scale analysis of C. albicans gene function. With the C. albicans genomic sequence, it is now possible to define the spectrum of gene function through analysis of strains that are defective in each gene. There has been no publicly available large-scale analysis of C. albicans gene function to date because C. albicans genetic analysis is cumbersome. We have developed a method that streamlines C. albicans gene disruption. A pilot set of 253 transposon-generated insertions into C. albicans ORFs has been used to identify new genes that govern pathogenicity and antifungal drug resistance. In addition, our results suggest that 36 of the genes are essential, and may be prospective drug targets. Our findings illustrate that genetic determinants of virulence and viability may be identified efficiently and cost-effectively through genetic analysis in C. albicans, rather than by inference from model yeasts that are seldom pathogens. We propose to expand the analysis through creation of a large set of cloned, sequenced C. albicans orf::Tn7-UAU1 insertion plasmids, construction of C. albicans orf::Tn7-UAU1/orf::Tn7-URA3 viable insertion homozygotes, and screening among the strains for alterations of phenotypes associated with pathogenicity, including filamentation, biofilm formation, antifungal drug resistance, host-cell interaction, and cell surface properties. Definition of the genes that govern these processes will lead to mechanistic insight, to candidate genes for mechanistic analysis, to therapeutic targets for pharmacological exploitation, and to rapid tests for the potential of an infecting strain to develop drug resistance.